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. 2003 Sep 30;100(20):11373-7.
doi: 10.1073/pnas.2034851100. Epub 2003 Sep 18.

Supercoiling and denaturation in Gal repressor/heat unstable nucleoid protein (HU)-mediated DNA looping

Affiliations

Supercoiling and denaturation in Gal repressor/heat unstable nucleoid protein (HU)-mediated DNA looping

Giuseppe Lia et al. Proc Natl Acad Sci U S A. .

Abstract

The overall topology of DNA profoundly influences the regulation of transcription and is determined by DNA flexibility as well as the binding of proteins that induce DNA torsion, distortion, and/or looping. Gal repressor (GalR) is thought to repress transcription from the two promoters of the gal operon of Escherichia coli by forming a DNA loop of approximately 40 nm of DNA that encompasses the promoters. Associated evidence of a topological regulatory mechanism of the transcription repression is the requirement for a supercoiled DNA template and the histone-like heat unstable nucleoid protein (HU). By using single-molecule manipulations to generate and finely tune tension in DNA molecules, we directly detected GalR/HU-mediated DNA looping and characterized its kinetics, thermodynamics, and supercoiling dependence. The factors required for gal DNA looping in single-molecule experiments (HU, GalR and DNA supercoiling) correspond exactly to those necessary for gal repression observed both in vitro and in vivo. Our single-molecule experiments revealed that negatively supercoiled DNA, under slight tension, denatured to facilitate GalR/HU-mediated DNA loop formation. Such topological intermediates may operate similarly in other multiprotein complexes of transcription, replication, and recombination.

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Figures

Fig. 1.
Fig. 1.
Experimental set-up. (a) A single DNA molecule tethering a magnetic bead to a surface can be twisted and stretched by using small magnets placed above the sample. DNA loop formation by GalR and HU reduces the bead-to-surface distance by an amount Δz at the expense of the work, FΔz, performed against the stretching force F. The tension on the DNA may be used to tune the transition rates, (τunlooped)–1 and (τlooped)–1, between the unlooped and looped state. (b) A typical telegraph-like signal. (c) A diagram illustrating the variation of ΔG for the reaction involving the DNA conformational change associated with loop opening. The activation energy for loop opening, Eb – δF, is slightly decreased on pulling, whereas the activation energy for loop formation, Ef + FΔz, is increased on pulling.
Fig. 2.
Fig. 2.
Traces of DNA length vs. time. The green dots are raw data, whereas the continuous colored lines are the averaged signal. The pertinent conditions are indicated beside each trace. Measurements were conducted at room temperature by using a solution containing 20 mM Tris·HCl (pH 7.8), 1 mM DTT, 50 mM NaCl, and 5 mM MgCl2. The GalR and HU concentrations were 25 nM and 50 nM, respectively. BSA and SSB were used at concentrations of 40 nM. (a) Variations in DNA extension at constant supercoiling (σ =–0.03) and force at F = 0.88 pN, unless otherwise stated. DNA with: no proteins (turquoise), GalR and HU (yellow), GalR and HU with a tension of 1.05 pN (purple), GalR and HU with a tension of 1.32 pN (cyan), GalR and HU and galactose (red), and GalR, HU, and SSB (dark green). (b) DNA extension at F = 0.88 pN, in the presence of GalR and HU as a function of supercoiling. σ = +0.03 (blue); σ = 0 (yellow); σ = –0.015 (purple); σ = –0.03 (cyan); and σ = –0.06 (red). σ is the superhelical density in the DNA defined as (LkLk0)/Lk0, where Lk is the linking number of DNA and is given by the sum of its twist (Tw) and writhe (Wr). In relaxed DNA, Lk = Lk0 = Tw = (number of bp)/10.4 bp. Similarly, in our single-molecule experimental conditions in which DNA molecules are stretched, the distribution between Tw and Wr is about 3:1 whereas in plasmids (unnicked and under no tension) it is about 1:3. As a result, the torsion within each molecule in our measurements at σ = –0.03 (i.e., with Tw ≈ 0.022 Lk0) is slightly higher than the torsion present in a plasmid at sigma = –0.06 (i.e., with Tw ≈ 0.015 Lk0).
Fig. 3.
Fig. 3.
Mean lifetimes of the looped and the unlooped DNA conformations were calculated from histograms of the lifetimes at F = 0.88 pN and 3% negative supercoiling.
Fig. 4.
Fig. 4.
Suggested model of the mechanism of GalR/HU-induced DNA loop formation. Negative supercoiling favors the opening of a bubble of a few base pairs in the DNA. HU binds preferentially to it, subsequently bending the ssDNA and stabilizing the interaction between two GalR dimers and loop closure.
Fig. 5.
Fig. 5.
In vitro transcription assay. Radiolabeled RNA products of an in vitro transcription assay were separated by gel electrophoresis (Left). A scheme of the procedure is on the Right. SSB added to lanes 3–5 did not interfere with repression of transcription from promoters P1 and P2 due to looping induced by GalR/HU (lane 2). No repression is observed without GalR/HU (lane 1).

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